1. KCNQ Potassium Channels
Malfunction in ion channels, due to mutations in genes encoding channel proteins or the presence of autoantibodies, are increasingly being implicated in causing disease conditions, termed channelopathies. For instance, dysfunction of potassium channels has been associated with the pathophysiology of a number of neurological disorders both affecting the central and peripheral nervous system (e.g., episodic ataxia, epilepsy, neuromyotonia, Parkinson's disease, congenital deafness, long QT syndrome). Potassium channels, which demonstrate a high degree of diversity and ubiquity, are fundamental in the control of membrane depolarisation and cell excitability. A common feature of potassium channelopathies is a reduction or loss of membrane potential repolarisation. Marketed potassium channels openers include for example flupirtine, an analgesic drug used for treating pain.
KCNQ polypeptides belong to the potassium channel family. KCNQ polypeptides associate to form homomeric or heteromeric potassium channels, each polypeptide corresponding to a subunit of the channel. Currently, five different members of the KCNQ family are known: KCNQ1, KCNQ2, KCNQ3, KCNQ4 and KCNQ5. Heteromeric KCNQ potassium channels can be comprised either of different members of the KCNQ family, or of KCNQ polypeptides associated with other members of the potassium channel family. KCNQ potassium channels underlie the M-current, an important regulator of neuronal excitability. Both their amino-terminal and their carboxyl-terminal extremities are located on the intracellular side of the membrane. These extremities play an important role both in interactions with other proteins and in modulation of the channel's activity.
KCNQ1 is expressed in heart, cochlea, intestine and kidney. It assembles with either the product of the KCNE1 gene or with the product of the KCNE3 gene. Mutations in the KCNQ1 gene have been shown to cause one form of inherited long QT syndrome and a form of deafness.
KCNQ2 was first cloned in 1996. In 1998, geneticists discovered that an inherited form of juvenile epilepsy, the benign familial neonatal convulsions, is caused by mutations in the potassium channel KCNQ2 (Singh et al. Nat Genet, 1998, 18:25-9; Biervert et al., Science, 1998, 279:403-6). More specifically, Bievert et al. showed that a five-base pair insertion deleting more than 300 amino acids from the carboxyl-terminus of KCNQ2 leads to impairment of potassium-selective currents in vitro. It was thus demonstrated that loss of function mutations in KCNQ2 causes the epileptic syndrome. Wang et al. showed KCNQ2 to be expressed in brain, and to be associated with KCNQ3. In addition, they showed that the KCNQ2/3 heteromultimers underlie the M-current (Wang et al., Science, 1998, 282:1890-3). In 2000, Main et al. showed that KCNQ2 is the molecular target of retigabine, a potent anticonvulsant compound, and that retigabine acts as a KCNQ2/3 potassium channel opener (Mol Pharmacol, 2000, 58:253-62). Biervert et al. determined that the KCNQ2 gene has at least 18 exons, occupying more than 50 kb of genomic DNA (Genet., 1999, 104:234-240). Until now six different isoforms of KCNQ2 produced by alternative splicing have been described (see, e.g., SwissProt Accession No. O43526).
KCNQ4 is expressed in inner ear, and it has been shown that mutation in the KCNQ4 gene lead to a form of inherited deafness.
KCNQ5 is expressed in brain and skeletal muscle, and can co-assemble with KCNQ3, suggesting that it may also play a role in the M-current heterogeneity. It has been suggested that KCNQ5 deficiency leads to retinal degeneration.
The activity of KCNQ channels has been shown to be modulated by Protein kinase A (PKA) and by the c-Src tyrosine kinase (Src). Schroeder et al. showed that currents generated by heteromeric KCNQ2/KCNQ3 channels can be increased by intracellular cyclic AMP, and that this effect is mediated by PKA. PKA stimulated current intensity by 66% (Schroeder et al., Epilepsia (2000) 41:1068-1069). Gamper et al. showed that coexpression of Src with KCNQ2/KCNQ3 heteromeric channels resulted in a 4.5-fold reduction of current density and a 2-fold slowing of activation kinetics at 0 mV. However, Src had no effect on currents generated by KCNQ2 homomultimeric channels (J. Neurosci. (2003) 23:84-95). In view of these results, modulation of KCNQ channels by kinases and phosphatases is believed to be important for control of neuronal excitability.
Studying KCNQ channels in humans and animal models is of great importance for the understanding of how M-channels control excitability at the cellular, network, and behavioral levels. A better understanding of the physiological role of KCNQ channels is a promising way of finding of new targets for novel diseases, thus leading to the possibility of novel screenings of drug candidates.
2. The PP2A Phosphatase
The PP2A phosphatase is an intracellular serine/threonine protein phosphatase constituted by two or three subunits. PP2A phosphatases comprise of a catalytic subunit (PP2A/C), a scaffolding subunit (PP2A/A) and eventually a regulatory subunit (PP2A/B).
Regulatory subunits are thought to confer tissue specificity, subcellular localization and developmental regulation to PP2A. More than eleven ndifferent regulatory subunits are currently known, and PP2A/Bγ is one of them. PP2A/Bγ is encoded by the PPP2R2C gene that was mapped to human chromosome 4p16 between markers D4S2925 and D4S3007 (Hu et al., Genomics., 2000, 67:83-6). The PP2A/Bγ protein can only be detected in brain and is enriched in the cytosolic fraction of the cell. Furthermore, PPP2R2C is developmentally regulated.
3. Mental Disorders
Mental disorders encompass a wide range of CNS disorders. Mental disorders include, e.g., mood disorders, psychotic disorders, anxiety disorders, childhood disorders, eating disorders and personality disorders, all these terms being defined according to the DSM-IV classification (Diagnosis and Statistical Manual of Mental Disorders, Fourth Edition, American Psychiatric Association, Washington D.C., 1994). Mood Disorders encompass bipolar I disorder (mania with or without major depression), bipolar II disorder (hypomania with major depression), cyclothymic disorder (numerous brief episodes of hypomania and minor depression), dysthymic disorder (prolonged minor depression without mania/hypomania) and major depressive disorder (major depression without mania). Psychotic disorders encompass schizophrenia, schizoaffective disorder, schizophreniform disorder, brief psychotic disorder, delusional disorder and shared psychotic disorder. Bipolar disorder, schizophrenia and depression are three particularly serious and widespread mental disorders.
3.1. Bipolar Disorder
Bipolar disorders are relatively common disorders, occurring in about 1.3% of the population, and have been reported to constitute about half of the mood disorders seen in psychiatric clinics with severe and potentially disabling effects. Bipolar disorders have been found to vary with gender depending of the type of disorder; for example, bipolar disorder I is found equally among men and women, while bipolar disorder II is reportedly more common in women. The age of onset of bipolar disorders is typically in the teenage years and diagnosis is typically made in the patient's early twenties. Bipolar disorders also occur among the elderly, generally as a result of a neurological disorder or other medical conditions. In addition to the severe effects on patients' social development, suicide completion rates among bipolar patients are reported to be about 15%.
Bipolar disorders are characterized by phases of excitement and often depression; the excitement phases, referred to as mania or hypomania, and depressive phases can alternate or occur in various admixtures, and can occur to different degrees of severity and over varying duration. Since bipolar disorders can exist in different forms and display different symptoms, the classification of bipolar disorder has been the subject of extensive studies resulting in the definition of bipolar disorder subtypes and widening of the overall concept to include patients previously thought to be suffering from different disorders. Bipolar disorders often share certain clinical signs, symptoms, treatments and neurobiological features with psychotic illnesses in general and therefore present a challenge to the psychiatrist to make an accurate diagnosis. Furthermore, because the course of bipolar disorders and various mood and psychotic disorders can differ greatly, it is critical to characterize the illness as early as possible in order to offer means to manage the illness over a long term.
The mania associated with the disease impairs performance and causes psychosis, and often results in hospitalization. This disease places a heavy burden on the patient's family and relatives, both in terms of the direct and indirect costs involved and the social stigma associated with the illness, sometimes over generations. Such stigma often leads to isolation and neglect. Furthermore, the earlier the onset, the more severe are the effects of interrupted education and social development.
The DSM-IV classification of bipolar disorder distinguishes among four types of disorders based on the degree and duration of mania or hypomania as well as two types of disorders which are evident typically with medical conditions or their treatments, or to substance abuse. Mania is recognized by elevated, expansive or irritable mood as well as by distractability, impulsive behavior, increased activity, grandiosity, elation, racing thoughts, and pressured speech. Of the four types of bipolar disorder characterized by the particular degree and duration of mania, DSM-IV includes:                bipolar disorder I, including patients displaying mania for at least one week;        bipolar disorder II, including patients displaying hypomania for at least 4 days, characterized by milder symptoms of excitement than mania, who have not previously displayed mania, and have previously suffered from episodes of major depression;        bipolar disorder not otherwise specified (NOS), including patients otherwise displaying features of bipolar disorder II but not meeting the 4 day duration for the excitement phase, or who display hypomania without an episode of major depression; and        cyclothymia, including patients who show numerous manic and depressive symptoms that do not meet the criteria for hypomania or major depression, but which are displayed for over two years without a symptom-free interval of more than two months.        
The remaining two types of bipolar disorder as classified in DSM-VI are disorders evident or caused by various medical disorder and their treatments, and disorders involving or related to substance abuse. Medical disorders which can cause bipolar disorders typically include endocrine disorders and cerebrovascular injuries, and medical treatments causing bipolar disorder are known to include glucocorticoids and the abuse of stimulants. The disorder associated with the use or abuse of a substance is referred to as “substance induced mood disorder with manic or mixed features”.
Evidence from twin and adoption studies, and the lack of variation in incidence worldwide, indicate that bipolar disorder is primarily a genetic condition, although environmental risk factors are also involved at some level as necessary, sufficient, or interactive causes. Aggregation of bipolar disorder and schizophrenia in families suggests that these two distinct disorders share some common genetic susceptibility. Several linkage studies of bipolar disorder have been reported, and several susceptibility regions have been identified. The regions that are associated with bipolar disorder include 1q31-q32, 4p16, 7q31, 12q23-q24, 13q32, 18p11.2, 21q22 and 22q11-q13 (Detera-Wadleigh et al. (1999) Proc Natl Acad Sci USA A96(10):5604-9). Some of these regions, like 4p16, 12q24, 18p11, 21q21 and 22q11 have been repeatedly implicated by independent investigators. Furthermore, some regions that are linked to bipolar disorder such as, e.g., 13q32 and 18p11.2, are also implicated in genome scans of schizophrenia, confirming that these two distinct disorders share some common genetic susceptibility. However, the genes underlying bipolar disorder and/or schizophrenia have not yet been identified.
3.2. Schizophrenia
There are an estimated 45 million people with schizophrenia in the world, with more than 33 million of them in the developing countries. In developed countries schizophrenia occurs in approximately 1% of the adult population at some point during their lives. If there is one grandparent with schizophrenia, the risk of getting the illness increases to about 3%; one parent with Schizophrenia, to about 10%. When both parents have schizophrenia, the risk rises to approximately 40%. Most schizophrenia patients are never able to work. Standardized mortality ratios (SMRs) for schizophrenic patients are estimated to be two to four times higher than the general population and their life expectancy overall is 20% shorter than for the general population. The most common cause of death among schizophrenic patients is suicide (in 10% of patients) which represents a 20 times higher risk than for the general population. Deaths from heart disease and from diseases of the respiratory and digestive system are also increased among schizophrenic patients.
Schizophrenia comprises a group of psychoses with either ‘positive’ or ‘negative’ symptoms. Positive symptoms consist of hallucinations, delusions and disorders of thought; negative symptoms include emotional flattening, lack of volition and a decrease in motor activity.
A number of biochemical abnormalities have been identified and, in consequence, several neurotransmitter based hypotheses have been advanced over recent years; the most popular one has been “the dopamine hypothesis,” one variant of which states that there is over-activity of the mesolimbic dopamine pathways at the level of the D2 receptor. However, researchers have been unable to consistently find an association between various receptors of the dopaminergic system and schizophrenia.
3.3. Depression
Depression is a serious medical illness that affects 340 million people worldwide. In contrast to the normal emotional experiences of sadness, loss, or passing mood states, clinical depression is persistent and can interfere significantly with an individual's ability to function. As a result, depression is the leading cause of disability throughout the world.
Symptoms of depression include depressed mood, diminished interest or pleasure in activities, change in appetite or weight, insomnia or hypersomnia, psycho-motor agitation or retardation, fatigue or loss of energy, feelings of worthlessness or excessive guilt, anxiety, inability to concentrate or act decisively, and recurrent thoughts of death or suicide. A diagnosis of unipolar major depression (or major depressive disorder) is made if a person has five or more of these symptoms and impairment in usual functioning nearly every day during the same two-week period. The onset of depression generally begins in late adolescence or early adult life; however, recent evidence suggests depression may be occurring earlier in life in people born in the past thirty years.
The World Health Organization predicts that by the year 2020 depression will be the greatest burden of ill-health to people in the developing world, and that by then depression will be the second largest cause of death and disability. Beyond the almost unbearable misery it causes, the big risk in major depression is suicide. Within five years of suffering a major depression, an estimated 25% of sufferers try to kill themselves. In addition, depression is a frequent and serious complication of heart attack, stroke, diabetes, and cancer. According to one recent study that covered a 13-year period, individuals with a history of major depression were four times as likely to suffer a heart attack compared to people without such a history. Depression may also be a feature in up to 50% of patients with mental disorders such as Parkinson's disease and Alzheimer's disease.
3.4. Treatment
There are currently no cures for mental disorders such as bipolar disorder, schizophrenia, depression and other mood disorders, so the objective of treatment is to reduce the severity of the symptoms, if possible to the point of remission. Due to the similarities in symptoms, schizophrenia, depression and bipolar disorder are often treated with some of the same medicaments.
3.4.1. Treatment of Bipolar Disorder
Depressive episodes may be treated like depression. However, most antidepressants can cause swings from depression to hypomania or mania and sometimes cause rapid cycling between them. Therefore, these drugs are used for only short periods, and their effect on mood is closely monitored. At the first sign of a swing to hypomania or mania, the antidepressant is stopped. Most people with manic-depressive disorder are given drugs with a mood-stabilizing effect such as lithium, carbamazepine and divalproex.
Lithium has no effect on normal mood but reduces the tendency toward mood swings in about 70% of the people with manic-depressive illness. A doctor monitors the level of lithium in the blood with blood tests. Possible adverse effects of lithium include tremor, muscle twitching, nausea, vomiting, diarrhea, thirst, excessive urination, and weight gain. Lithium can make acne or psoriasis worse, can cause the blood levels of thyroid hormone to fall, and rarely can cause excessive urination. A very high level of lithium in the blood can cause a persistent headache, mental confusion, drowsiness, seizures, and abnormal heart rhythms. Adverse effects are more likely to occur in the elderly. Women who are trying to become pregnant must stop taking lithium, because lithium may cause heart defects in a developing fetus.
Newer drug treatments have evolved over the past several years. These include the carbamazepine and divalproex. However, carbamazepine can seriously reduce the number of red and white blood cells, and divalproex can cause liver damage (primarily in children). With careful monitoring by a doctor, these problems are rare, and carbamazepine and divalproex are useful alternatives to lithium, especially for people with the mixed or rapid cycling form of manic-depressive illness who haven't responded to other treatments.
3.4.2. Treatment of Schizophrenia
For schizophrenia, antipsychotic medications are the most common and most valuable treatments. There are four main classes of antipsychotic drugs which are commonly prescribed for schizophrenia. The first, neuroleptics, exemplified by chlorpromazine (Thorazine), has revolutionized the treatment of schizophrenic patients by reducing positive (psychotic) symptoms and preventing their recurrence. Patients receiving chlorpromazine have been able to leave mental hospitals and live in community programs or their own homes. But these drugs are far from ideal. Some 20% to 30% of patients do not respond to them at all, and others eventually relapse. These drugs were named neuroleptics because they produce serious neurological side effects, including rigidity and tremors in the arms and legs, muscle spasms, abnormal body movements, and akathisia (restless pacing and fidgeting). These side effects are so troublesome that many patients simply refuse to take the drugs. Besides, neuroleptics do not improve the so-called negative symptoms of schizophrenia and the side effects may even exacerbate these symptoms. Thus, despite the clear beneficial effects of neuroleptics, even some patients who have a good short-term response will ultimately deteriorate in overall functioning.
The well known deficiencies in the standard neuroleptics have stimulated a search for new treatments and have led to a new class of drugs termed atypical neuroleptics. The first atypical neuroleptic, Clozapine, is effective for about one third of patients who do not respond to standard neuroleptics. It seems to reduce negative as well as positive symptoms, or at least exacerbates negative symptoms less than standard neuroleptics do. Moreover, it has beneficial effects on overall functioning and may reduce the chance of suicide in schizophrenic patients. It does not produce the troubling neurological symptoms of the standard neuroleptics, or raise blood levels of the hormone prolactin, excess of which may cause menstrual irregularities and infertility in women, impotence or breast enlargement in men. Many patients who cannot tolerate standard neuroleptics have been able to take clozapine. However, clozapine has serious limitations. It was originally withdrawn from the market because it can cause agranulocytosis, a potentially lethal inability to produce white blood cells. Agranulocytosis remains a threat that requires careful monitoring and periodic blood tests. Clozapine can also cause seizures and other disturbing side effects (e.g., drowsiness, lowered blood pressure, drooling, bed-wetting, and weight gain). Thus only patients who do not respond to other drugs usually take Clozapine.
Researchers have developed a third class of antipsychotic drugs that have the virtues of clozapine without its defects. One of these drugs is risperidone (Risperdal). Early studies suggest that it is as effective as standard neuroleptic drugs for positive symptoms and may be somewhat more effective for negative symptoms. It produces more neurological side effects than clozapine but fewer than standard neuroleptics. However, it raises prolactin levels. Risperidone is now prescribed for a broad range of psychotic patients, and many clinicians seem to use it before clozapine for patients who do not respond to standard drugs, because they regard it as safer. Another new drug is Olanzapine (Zyprexa) which is at least as effective as standard drugs for positive symptoms and more effective for negative symptoms. It has few neurological side effects at ordinary clinical doses, and it does not significantly raise prolactin levels. Although it does not produce most of clozapine's most troubling side effects, including agranulocytosis, some patients taking olanzapine may become sedated or dizzy, develop dry mouth, or gain weight. In rare cases, liver function tests become transiently abnormal.
3.4.3. Treatment of Depression
Several types of antidepressants are available. These antidepressants belong to four main categories: tricyclic antidepressants, selective serotonin reuptake inhibitors, monoamine oxidase inhibitors and psychostimulants. Tricyclic antidepressants include, e.g., Amitriptyline, Amoxapine, Bupropion, Clomipramine, Desipramine, Doxepin, Imipramine, Maprotiline, Nefazodone, Nortriptyline, Protriptyline, Trazodone, Trimipramine and Venlafaxine. Selective serotonin reuptake inhibitors include, e.g., Fluoxetine, Fluvoxamine, Paroxetine and Sertraline. Monoamine oxidase inhibitors include, e.g., Isocarboxazid, Pargyline, Phenelzine and Tranylcypromine. Psychostimulants include, e.g., Dextroamphetamine and Methylphenidate.
All these antidepressants must be taken regularly for at least several weeks before they begin to work. The chances that any given antidepressant will work for a particular person are about 65%. However, most of these drugs have side effects varying with each type of drug. For example, the tricyclic antidepressants often cause sedation and lead to weight gain. They can also be associated with side effects such as an increased heart rate, a decrease in blood pressure when the person stands or blurred vision.
Thus, for mental disorders such as bipolar disorder, schizophrenia, depression and other mood disorders, known molecules used for the treatment have side effects and act only against the symptoms of the disease. Consequently, there is a strong need for new molecules without associated side effects that are specifically directed against targets which are involved in the causal mechanisms of such mental disorders. Therefore, there is a need to identify proteins involved in bipolar disorder and schizophrenia. Providing new targets involved in bipolar disorder and schizophrenia will allow new screenings for drugs, resulting in new drugs that are efficient in treatment of these serious mental disorders.
Furthermore, there is also a need for diagnostic tools. There is increasing evidence that leaving schizophrenia untreated for long periods early in course of the illness may negatively affect the outcome. However, the use of drugs is often delayed for patients experiencing a first episode of the illness. The patients may not realize that they are ill, or they may be afraid to seek help; family members sometimes hope the problem will simply disappear or cannot persuade the patient to seek treatment; clinicians may hesitate to prescribe antipsychotic medications when the diagnosis is uncertain because of potential side effects. Indeed, at the first manifestation of the disease, schizophrenia or bipolar disorder is difficult to distinguish from, e.g., drug-related disorders and stress-related disorders. Accordingly, there is a need for new methods for detecting a susceptibility to mental disorders such as bipolar disorder, schizophrenia, and depression.